PROJECT SUMMARY The holy grail of structural biology, i.e., the simultaneous quantitative determination of a protein?s structure and function, remains very difficult to attain. This application pertains to the development of a new, cutting-edge optical approach that allows the resolution of sub-nanometer-scale distances and distance changes in real time: distance-resolving Voltage Clamp Fluorometry (drVCF). drVCF combines the use of small, spectrally-identical, Cys-attached fluorophores of variable length with Trp-induced collisional quenching. Crucially, fluorophore range and flexibility are accounted for by radial probability density functions (pdfs) generated by fluorophore molecular dynamics (MD) simulations. The pdfs are used to simultaneously fit the optical signals of multiple labels and obtain highly constrained distance information immediately relevant to protein structure (from the Trp side-chain to the labeled Cys C? atom). drVCF encompasses the benefits of other optical structural approaches (FRET, LRET, etc.), such as wide applicability and physiologically-relevant experimental conditions; but also distinct advantages, such as (i) the ability to measure intramolecular distances and functionally-relevant distance changes with a very fine grain (<2 measurement error in a preliminary evaluation), practically excluding intersubunit and intermolecular signal contamination (<2.2 nm range); (ii) the acquisition of structural data in real time, allowing the simultaneous tracking of structure and the kinetics of structural change; (iii) the ability to acquire data from conducting channels without large protein adjuncts such as toxin-mounted fluorophores or large fluorescent proteins; (iv) no dependence on fluorophore dipole orientation. As all scientific approaches, drVCF carries assumptions and limitations. In this proposal, the capabilities and limitations of drVCF will be evaluated in established models of structural biology, over three Specific Aims. Aim 1: Validate a New Optical Approach to Measure Functionally-relevant Intramolecular Protein Distances (drVCF) Using Rigid Rod-like Peptides of Known Length. As Stryer and Haugland did to calibrate FRET, drVCF accuracy will be evaluated by measuring the length of rigid polyproline peptides. Aim 2: Validate drVCF in a Well Characterized Soluble Protein of Known Structure. drVCF will be used to measure intramolecular distances in T4 lysozyme, a gold standard in structural biology, to evaluate the applicability of this approach in proteins and its accuracy. Aim 3: Validate drVCF in a Voltage-sensitive Membrane Protein with Known Resting/Active Structures. The voltage-sensing domain of the voltage sensitive phosphatase (Ci-VSP) was recently crystallized in the Resting and Active states. drVCF will be combined with cut-open oocyte voltage clamp to test its ability to measure voltage-dependent distance changes. This approach was developed following highly-encouraging preliminary experiments. The proposed studies may reveal practical pitfalls and limitations, but with them also the opportunity to rectify and refine this highly innovative and potentially ground breaking approach.